N-methyl-D-aspartate receptor subunits are non-myosin targets of myosin regulatory light chain.
Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the "GXXXR" portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.
Bajaj
Gaurav
G
Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.
Zhang
Yong
Y
Schimerlik
Michael I
MI
Hau
Andrew M
AM
Yang
Jing
J
Filtz
Theresa M
TM
Kioussi
Chrissa
C
Ishmael
Jane E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism*; D024462:Myosin Type II / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D002147:Calmodulin; D008274:Magnesium; D018994:Myosin Light Chains; D024462:Myosin Type II; D020033:Protein Isoforms; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; C121001:NR2B NMDA receptor